Project Summary Tracking the evolution of drug resistance for highly potent HIV protease inhibitors Project 2 - Swanstrom, UNC, Chapel Hill HIV-1 protease inhibitors are unique among all HIV-1 inhibitors in that they are transition state analogs which allows for binding constants that are orders of magnitude greater than any other type of inhibitor. Resistance to any inhibitor reflects the sequential addition of single amino acid changes that allow sufficient replication and resistance to the inhibitor to favor replication over the wild type virus in the presence of the inhibitor. However, if the resistance contribution of the mutation is too low to confer a growth advantage at the given level of drug compared to the wild type then resistance will not evolve. Also, potent inhibitors can restrict replication to such an extent that the evolution of resistance is slowed or even stopped. Protease inhibitors, because of their tight binding properties, offer the only possible inhibitor that can be envisioned to be able to avoid resistance and to drive (or maintain) the population at levels of negligible replication as a single agent. For this reason we propose this proof-of-concept study to explore the nature of resistance to these very tight-binding inhibitors using three approaches to identifying resistance mutations: selection of virus for resistance in expanded cell culture conditions; selection of virus for resistance in a humanized mouse model; and screening of large, genetically complex populations of protease expressed in yeast in the presence of protease inhibitors. We will also determine if these very tight binding inhibitors are able to suppress viral replication in the humanized mouse model as single agents. Finally, we will examine the role of mutations outside of the protease, in Gag, RT/IN, and gp41 to determine if there are mutations that affect fitness beyond the known mutations in the protease cleavage site.